This application proposes continuation of a project designed to elucidate the mechanisms that control ischemic retinopathy. We have been studying the role of the superoxide generating enzyme NADPH oxidase in triggering over-expression of vascular endothelial growth factor (VEGF) resulting in vascular inflammatory reactions and pathological angiogenesis. Our research with models of oxygen-induced retinopathy (OIR), diabetic retinopathy (DR) and endotoxin-induced uveitis (EIU) has revealed that the NADPH oxidase NOX2 isoform has a key role in inducing production of reactive oxygen species (ROS) and angiogenic cytokines and causing vascular inflammatory reactions and vitreo-retinal neovascularization. We now propose to determine the specific role of the urea/ornithine pathway enzyme arginase in these effects. Our studies in models of diabetes have shown that increases in arginase activity cause vascular endothelial cell (EC) dysfunction by decreasing availability of L-arginine to EC nitric oxide synthase (eNOS) which decreases NO and increases ROS. Increasing arginase activity may also contribute to pathological retinal angiogenesis and fibrosis/gliosis by increasing formation of polyamines and proline, which induce cell growth and collagen production, respectively. Our preliminary data show that arginase activity and arginase I (AI) expression are increased in models of OIR, DR or EIU and that the increase in AI is abrogated in mice that lack NOX2. Our studies also indicate that increases in arginase activity and AI expression are correlated with increased cytokine expression increased ROS formation and retinal vascular dysfunction. Furthermore, deletion of one copy of the AI gene in combination with AII deletion decreases cytokine production in both DR and EIU. Moreover, preliminary studies in the OIR model indicate that treatment with an arginase inhibitor reduces vitreoretinal angiogenesis and enhances physiological angiogenesis. Based on our previous work and new preliminary data, our global hypothesis is that NADPH oxidase-induced activation of the arginase pathway has a key role in causing retinal vascular dysfunction and inducing pathological angiogenesis and fibrosis/gliosis during retinopathy. Our specific aims are as follows: 1. Determine the role of NADPH oxidase-derived ROS in increasing arginase expression/activity and uncoupling NOS during retinopathy. 2. Define the molecular mechanisms of arginase activation during retinopathy. 3. Test whether diabetes causes retinal vascular dysfunction by activating arginase. 4. Test whether retinal ischemia causes pathological angiogenesis and fibrosis/gliosis by increasing arginase. PUBLIC HEALTH RELEVANCE: Retinal vascular disease and pathological retinal angiogenesis are a leading cause of blindness in working aged adults and the elderly. Blinding retinal diseases that involve pathological angiogenesis all follow the same pathological progression, beginning with vascular inflammatory reactions and dysfunction and injury of the vascular endothelial cell lining and progressing to pathological angiogenesis, followed by fibrosis/gliosis and retinal detachment. Understanding the factors and sequence of events which initiate this process is critical to preventing and treating retinal vascular diseases. Limiting the actions of an enzyme which can cause inflammatory reactions and vascular endothelial cell damage and prevent the retina from getting enough blood offers much promise for reducing the incidence of this blinding condition.